key: cord-0279376-3dglk6rz
authors: Huang, Yanqing; Garcia Garcia, Carolina J.; Lin, Daniel; Nguyen, Nicholas D.; Fujimoto, Tara N.; Zhao, Jun; Lee, Jaewon J.; Bernard, Vincent; Yu, Meifang; Delahoussaye, Abagail M.; Phan, Jae L.; Deorukhkar, Amit; Molkentine, Jessica M.; Fuentes, Natividad R.; Turner, Madeleine C.; Saur, Dieter; Maitra, Anirban; Taniguchi, Cullen M.
title: Stromal HIF2 Regulates Immune Suppression in the Pancreatic Cancer Microenvironment
date: 2021-05-22
journal: bioRxiv
DOI: 10.1101/2021.05.21.445190
sha: 19a2a5909a86ea76358f4760927f3bc9a41668a0
doc_id: 279376
cord_uid: 3dglk6rz

Background & Aims Pancreatic ductal adenocarcinoma (PDAC) has a hypoxic, immunosuppressive stroma, which contributes to its resistance to immune checkpoint blockade therapies. The hypoxia-inducible factors (HIFs) mediate the cellular response to hypoxia, but their role within the PDAC tumor microenvironment remains unknown. Methods We used a dual recombinase mouse model to delete Hif1α or Hif2α in α-smooth muscle actin (αSMA)-expressing cancer-associated fibroblasts (CAFs) arising within spontaneous pancreatic tumors. The effects of CAF-Hif2α expression on tumor progression and composition of the tumor microenvironment were evaluated by Kaplan-Meier analysis, quantitative real-time polymerase chain reaction, histology, immunostaining, and by both bulk and single-cell RNA sequencing. CAF-macrophage crosstalk was modeled ex vivo using conditioned media from CAFs after treatment with hypoxia and PT2399, a HIF2 inhibitor currently in clinical trials. Syngeneic flank and orthotopic PDAC models were used to assess whether HIF2 inhibition improves response to immune checkpoint blockade. Results CAF-specific deletion of HIF2, but not HIF1, suppressed PDAC tumor progression and growth, and improved survival of mice by 50% (n = 21-23 mice/group, Log-rank P = 0.0009). Deletion of CAF-HIF2 modestly reduced tumor fibrosis and significantly decreased the intratumoral recruitment of immunosuppressive M2 macrophages and regulatory T cells. Treatment with the clinical HIF2 inhibitor PT2399 significantly reduced in vitro macrophage chemotaxis and M2 polarization, and improved tumor responses to immunotherapy in both syngeneic PDAC mouse models. Conclusions Together, these data suggest that stromal HIF2 is an essential component of PDAC pathobiology and is a druggable therapeutic target that could relieve tumor microenvironment immunosuppression and enhance immune responses in this disease.

*Authors share co-first authorship. Data & Resource Sharing: Data, materials, and reagents will be made available 32 to other researchers upon request. 33

Pancreatic ductal adenocarcinoma (PDAC) responds poorly to most 70 cancer treatments, including immunotherapy 1 . This therapeutic recalcitrance may 71 stem from PDAC's extensive desmoplastic stroma, which suppresses anti-tumor 72 immunity 2 and increases intratumoral pressure 3 , resulting in severe hypoxia 4 and 73 impaired drug delivery 5 . Cancer-associated fibroblasts (CAFs) are the main 74 components and producers of stroma in PDAC 6 . Efforts to physically disrupt the 75 hypoxic stromal component through Sonic hedgehog protein inhibition 7 , selective 76 fibroblast depletion 8 , or recombinant human hyaluronidase 9 have effectively 77 lowered stromal content but paradoxically led to worse outcomes in both 78 preclinical studies and clinical trials. These data argue that the initially promising 79 strategy of physically ablating the PDAC stroma may be clinically 80 counterproductive, warranting a different approach. 81

The hypoxia-inducible factors 1 (HIF1) and 2 (HIF2) are stabilized in low 82 oxygen and have been hypothesized to mediate therapeutic resistance 10 and 83 aggressive growth of PDAC 11 . Deletion of HIF1 12 or HIF2 13 in the pancreatic 84 epithelial compartment failed to change overall survival in mice with spontaneous 85 PDAC. However, the function of HIFs in other prominent compartments of the 86 pancreatic tumor microenvironment (TME) remains unclear. Given the 87 importance of the tumor stroma in PDAC oncobiology, we investigated the role of 88 HIF signaling in CAFs and its impact on the PDAC TME. 89

Here, we elucidated the function of the HIFs within the PDAC stroma 90 using a dual recombinase model to spatiotemporally alter HIF1 or HIF2 signaling 91 only in activated fibroblasts reprogrammed within spontaneous murine pancreatic 92 tumors (also known as CAFs). We found that CAF-specific deletion of HIF2, but 93 not HIF1, improved survival from pancreatic cancer by reducing the recruitment 94 of immunosuppressive macrophages. We further showed that therapeutic HIF2 95 inhibition improved responses to immune checkpoint blockade, indicating this is a 96 potential combinatorial therapeutic strategy for PDAC. of Arg1 was also performed on RAW 264.7 murine macrophages treated with the 208 indicated conditions. Primers are listed in Supplementary Table 3 . week-old C57BL/6 male mice. After 2 weeks of recovery, murine aCTLA4 (clone 252 9D9, Merck) and murine aPD1 (muDX400, Merck) or isotype control were 253 administered IP every 4 days at 20 µg/mouse, 200 µg/mouse, and 220 254 µg/mouse, respectively for 2 weeks. PT2399 was administered 5 days per week, 255 twice daily for 3 weeks, at 50 mg/kg via oral gavage ( Figure 4I ). Tumor burden 256 was monitored by ultrasound. Mice were age-matched but group assignment was 257 unblinded. 258 259

Survival was analyzed by the Kaplan-Meier method and log-rank test. 261

Student's t test was used to analyze parametric data sets and the Mann-Whitney 262 U test was used for non-parametric data sets. All statistical analyses were 263 performed using GraphPad Prism V.8 (RRID:SCR_002798), with a significance 264 level of a = 0.05. 265

Results 267

We used a dual recombinase system to constrain the deletion of Hif1a or 269 

WT and KO groups. Immunohistochemical analysis confirmed a reduction of 286 HIF2 expression in CAFs within KPF tumors (Supplementary Figure 1C) . 287

Surprisingly, loss of stromal HIF1 had no effect on tumor growth or survival 288

(median survival, 91 days for KO versus 100 days for WT; Supplementary Figure  289 1D-E). 290

In contrast, HIF2 ablation in CAFs significantly decreased tumor growth 291 and improved survival (median survival, 120 days for KO versus 80 days for WT; 292 n = 21-23 mice/group, Log-rank P = 0.0009; Figure 1C ). Histological analyses of 293 the pancreata revealed well-differentiated PDAC foci in both groups, yet 294 remarkably, we found no gross or microscopic evidence of tumor tissue in the 295 sections analyzed from six of the HIF2-depleted mice, suggesting that deletion of 296 stromal HIF2 may also influence PDAC oncogenesis and/or progression 297 To evaluate this hypothesis, we established CAF and normal fibroblast 334 lines from spontaneous pancreatic tumors and normal pancreata, respectively. 335

Both cell lines were cultured in hypoxia to stabilize HIF2 and to approximate in 336 vivo TME conditions, and were then treated with either vehicle or the clinical 337 HIF2 inhibitor PT2399 22 . We found that conditioned media from hypoxic CAFs 338 stimulated macrophage migration in a HIF2-dependent fashion ( Figures 3A-B) . 

We performed single-cell RNA sequencing (scRNA-seq) to interrogate the 379 impact of CAF-specific HIF2 signaling on other cells in the PDAC TME. We 380 analyzed the transcriptomes from 22,635 single cells isolated from three KPF 381 Moreover, IHC staining of tumor sections for the Treg marker FoxP3 showed that 406 CAF-HIF2 KO tumors had significantly fewer Tregs than CAF-HIF2 WT tumors (n 407 = 5-6 tumors/group, P = 0.014; Figure 4F ). These data strongly suggest that 408 deletion of HIF2 in CAFs reduces the PDAC immunosuppressive landscape. 409 410

PDAC is highly resistant to immunotherapy 1 , but recent studies have 412 suggested that targeting non-redundant pathways by combining anti-CTLA4 and 413

anti-PD1 therapies may overcome the inherent TME immunosuppression 30 . 414

Since HIF2 deletion reduced the number of immunosuppressive M2-polarized 415

TAMs and Tregs, we reasoned that PT2399 might improve response to 416 checkpoint immunotherapy. To test this hypothesis, we implanted KPC cells 417 subcutaneously into syngeneic C57BL/6 mice and assigned them to one of four 418 treatments: vehicle plus IgG control, vehicle plus anti-CTLA4 antibody (aCTLA4), 419 PT2399 plus IgG, or PT2399 plus aCTLA4 ( Figure 4G ). We found that the 420 combination of PT2399 with aCTLA4 significantly slowed tumor growth (n = 10 421 mice/group, P = 0.038), while treatment with either drug alone had no discernible 422 effect ( Figure 4H ). 423

We next implanted KPC cells orthotopically into syngeneic C57BL/6 mice 424 to test whether HIF2 inhibition enhanced response to dual checkpoint blockade 425 (DCB) with aCTLA4 and anti-PD1 antibody (aPD1). Mice were assigned to one 426 of four treatments: vehicle plus IgG, vehicle plus DCB, PT2399 plus IgG, or 427 PT2399 plus DCB, with the goal to assess 60-day survival ( Figure 4I ). The 428 experiment was prematurely terminated due to institutional mandates related to 429 COVID-19, yet the survival rate at 45 days in mice that received combined 430 PT2399 and DCB was 100%, significantly better than the survival rate in the 431 groups treated with IgG control (n = 10 mice/group, P ≤ 0.0005), and trending 432 toward improved survival compared to mice treated with DCB and vehicle (n = 10 433 mice/group, P = 0.067; Figure 4J ). Taken together, these results suggest that 434 shifting the cellular composition of the TME, rather than by altering fibrosis, which 448 was unchanged in our model. We observed more TAMs and Tregs in tumors 449 from mice with intact HIF2 function compared to mice with HIF2 deletion in CAFs. 450

These data contrast with findings from a previous study in which depletion of 451 aSMA+ CAFs reduced fibrosis and increased Tregs and cancer progression 8 . 452

These phenotypic differences are most likely explained by the different 453 approaches of the two studies: ours targeted CAF functionality under hypoxia, 454 while the former study ablated CAFs altogether. We note that we used our dual We demonstrated with single-cell resolution that M2-polarized TAMs were 487 a major source of CD86 and PD-L1 in the TME, whereas their respective 488 receptors were expressed in a subset of T cells. Thus, we infer that these TAMs 

Multiomics Prediction of Response Rates to Therapies 540 to

Plays an Essential Role in the Accumulation and Modulation of Infiltrated 544

Immune Cells in Pancreatic Adenocarcinoma

Compression of pancreatic 547 tumor blood vessels by hyaluronan is caused by solid stress and not 548 interstitial fluid pressure

Pancreatic tumors show high levels of 550 hypoxia

Hyaluronan, fluid pressure, and stromal 552 resistance in pancreas cancer

Fibroblasts in Pancreatic Ductal 554

Phase 2 study of 557 vismodegib, a hedgehog inhibitor, combined with gemcitabine and nab-558 paclitaxel in patients with untreated metastatic pancreatic 559 adenocarcinoma

Depletion of 561 carcinoma-associated fibroblasts and fibrosis induces immunosuppression 562 and accelerates pancreas cancer with reduced survival

Why HALO 301 Failed and Implications 565 for Treatment of Pancreatic Cancer

Opportunities (and Problems) for Cancer Therapy

High nuclear hypoxia-inducible factor 570 1 alpha expression is a predictor of distant recurrence in patients with 571 resected pancreatic adenocarcinoma

Hif1a Deletion Reveals Pro-Neoplastic 574

Function of B Cells in Pancreatic Neoplasia

Wnt Signaling by HIF2α during Early Pancreatic Tumorigenesis

Uncoupling Cancer Mutations Reveals 580

Critical Timing of p53 Loss in Sarcomagenesis

Generation of primary tumors with 583

Flp recombinase in FRT-flanked p53 mice

A next-generation dual-586

recombinase system for time-and host-specific targeting of pancreatic 587 cancer

Efficient temporally-controlled 589 targeted mutagenesis in smooth muscle cells of the adult mouse

Enables Ablative Radiotherapy and Improves Survival in Unresectable 593

Protects Against Radiation-Induced Gastrointestinal Toxicity via HIF2

Both p16(Ink4a) and the p19

p53 pathway constrain progression of pancreatic adenocarcinoma in the 599 mouse

RSEM: accurate transcript quantification from RNA-Seq 601 data with or without a reference genome

Targeting renal cell carcinoma with a HIF-604 2 antagonist

Hypoxia-inducible Factor-1α Is a 606

Positive Factor in Solid Tumor Growth

Acute postnatal ablation of Hif-2α

Hedgehog-Mediated Tumor-Stromal Interactions in Pancreatic Cancer

Tumor-associated macrophages: from mechanisms to 614 therapy

Cancer-associated fibroblasts promote M2 616 polarization of macrophages in pancreatic ductal adenocarcinoma

Combination of anti-angiogenic therapy 619 and immune checkpoint blockade normalizes vascular-immune crosstalk 620 to potentiate cancer immunity

Targeting the HIF2-VEGF axis in renal cell 623 carcinoma

Radiation and dual 625 checkpoint blockade activate non-redundant immune mechanisms in 626 cancer

Cross-Species Single-Cell Analysis 628 of Pancreatic Ductal Adenocarcinoma Reveals Antigen-Presenting 629

Pancreatic stellate cells support 631 tumour metabolism through autophagic alanine secretion

Tumor microenvironment derived 634 exosomes pleiotropically modulate cancer cell metabolism

Hypoxia-inducible factor 2α 637 regulates macrophage function in mouse models of acute and tumor 638 inflammation

Stromal cues regulate the 640 pancreatic cancer epigenome and metabolome. Proceedings of the 641 National Academy of